The present invention is in the field of enzyme proteins that are related to the glutathione peroxidase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Many human enzymes serve as targets for the action of pharmaceutically active compounds. Several classes of human enzymes that serve as such targets include helicase, steroid esterase and sulfatase, convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase, decarboxylase, isomerase and reductase. It is therefore important in developing new pharmaceutical compounds to identify target enzyme proteins that can be put into high-throughput screening formats. The present invention advances the state of the art by providing novel human drug target enzymes related to the glutathione peroxidase subfamily.
Glutathione peroxidase (EC 1.11.1.9) catalyzes the reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides by reduced glutathione and functions in the protection of cells against oxidative damage. This enzyme, found mainly in the cytosol of mammalian cells, is unusual in its content of a selenocysteine residue in its active site that is encoded by a TGA opal codon (Chambers et al., 1986). The glutathione peroxidase found in plasma is immunologically distinct from the erythrocyte and liver cytosolic enzymes. It also has some differences in physical and kinetic properties. Takahashi et al. (1990) isolated cDNA clones coding for plasma GPX. They found that the nucleotide sequence consisted of a 678-bp open reading frame coding for a 226-amino acid polypeptide with a molecular mass of 25,389. The amino acid sequence showed only 44% homology with human cellular GPX. Northern blot analysis showed a single transcript of 2.2 kb in the polyadenylated RNA fractions of human placenta and of a human hepatic cell line, HepG2, but not in those of human liver and endothelial cells. Takahashi et al. (1990) concluded that as the plasma enzyme contains 1 atom of selenium per subunit, the in-frame TGA observed at positions 217-219 could be assigned to selenocysteine.
Chu et al. (1992) found that glutathione peroxidase-3 is expressed in kidney, lung, heart, breast, placenta, and, in the human but not the rodent, in liver as well. By Southern analysis of genomic DNA from human/hamster somatic cell hybrids, Chu (1994) mapped the GPX3 gene to chromosome 5.
The present invention has substantial similarity to human plasma glutathione peroxidase (GSHPx). HSHPx has been shown to be a selenium-containing enzyme immunologically distinct from cellular GSHPx. Nucleotide sequence analysis of the obtained clones revealed that GSHPx consisted of a 678-base pair open reading frame coding for a 226-amino acid polypeptide with a Mr of 25,389. About 50% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of the peptides in a lysine endopeptidase-digest of the purified enzyme. Northern blot analysis revealed a single transcript of 2.2 kilobases in the poly(A)+RNA fractions of human placenta and HepG2 (a human hepatic cell line), but not that of human liver and endothelial cells.
Tujebajeva et al., EMBO Rep 2000 Aug;1(2):158-63; Takahashi et al., J Biochem (Tokyo) August 1990; 108(2):145-8; Chambers et al., EMBO J. 5: 1221-1227, 1986; Chu et al., Cytogenet. Cell Genet. 66: 96-98, 1994; Chu et al., Blood 79: 3233-3238, 1992.
Enzyme proteins, particularly members of the glutathione peroxidase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of enzyme proteins. The present invention advances the state of the art by providing previously unidentified human enzyme proteins, and the polynucleotides encoding them, that have homology to members of the glutathione peroxidase subfamily. These novel compositions are useful in the diagnosis, prevention and treatment of biological processes associated with human diseases.
The present invention is based in part on the identification of amino acid sequences of human enzyme peptides and proteins that are related to the glutathione peroxidase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in the brain glioblastoma cell line, ovary adenocarcinoma cell line, panceras epitheloid carcinoma, uterus leiomyosarcoma, lung large cell carcinoma, fetal brain and human whole liver.